This article describes a method for identifying different subsets of peripheral blood mononuclear cells (PBMCs) using flow cytometry. The protocol includes the use of fluorescent viability dyes and antibody cocktails to label specific cell types.
Take peripheral blood mononuclear cells or PBMCs, a mixed-cell population including lymphocytes and monocytes.
Add a fluorescent viability dye to discriminate dead cells, and a fluorescently labeled antibody cocktail to identify different cell types.
The fluorochrome A-conjugated antibodies bind to surface markers — CD3 on T cells, γδ T cell receptor or TCR on γδ T cells, and CD56 on natural killer or NK cells.
The fluorochrome B-conjugated antibodies bind to CD4 and CD8 on T cells, CD19 on B cells, and CD14 on monocytes.
Using flow cytometry, identify the live cell subsets.
While CD8+ and CD4+ T cells are CD3+, CD4+ cells lie between CD3 single-positive and CD3-CD8 double-positive cells.
γδ T cells express γδ TCRs and higher CD3 compared to CD4+ and CD8+ cells.
B cells and NK cells are CD3- but are identified by CD19 on B cells and CD56 on NK cells.
Monocytes are identified by CD14 expression.
To prepare the PBMCs for staining, transfer 100 microliters of each sample to a 96-well V-bottom plate. Centrifuge the plate at 350 times g for three minutes at room temperature. Carefully, aspirate the supernatant without disturbing the cell pellet. To each well, add 100 microliters of PBS containing a live/dead fixable dye that reacts with free amine on proteins, and resuspend the PBMC mixture carefully.
Subsequently, allow the plate to sit for 10 minutes at room temperature for labeling of dead cells. For each sample, prepare 30 microliters of a mix containing all seven antibodies. At this stage, titrated antibodies against different target molecules and in different fluorochromes can be added as well. Centrifuge the 96-well plate at 350 times g for three minutes at room temperature, and carefully aspirate the supernatant without disturbing the cell pellet.
Add the antibody cocktail to each well, and resuspend carefully without generating bubbles. Incubate for 30 minutes at room temperature in the dark. After 30 minutes, add 150 microliters of staining buffer to each well, and centrifuge the plate at 350 times g for three minutes at room temperature. Carefully aspirate the supernatant without disturbing the cell pellet, and resuspend the cells in 200 microliters of PBS. The cells are now ready for flow cytometry analysis.
To begin this two-fluorochrome, seven-marker gating strategy, select the lymphocyte gate and create a dot-plot with one of the two fluorochromes used in this protocol on each axis. Gate on CD8-positive T cells, identified as CD3 and CD8 double-positive cells at the top-right corner of the dot-plot. Exclude the dim CD8 population, which might contain NK T cells. Gate on CD4-positive T cells, identified as the population in between CD8-positive T cells and the CD3 single-positive populations.
Gate on gamma-delta T cells, identified as high CD3 cells. Subdivide gamma-delta T cells to CD8-positive and CD8-negative. Gate on NK cells, identified as the population in between CD3-positive and CD3-negative cells. Subdivide the NK cells to CD8-positive and CD8-negative Gate on B cells, identified as the CD3-negative, CD19-positive population on the lower right corner of the dot-plot.
Select the monocyte gate and create a dot-plot with one of the two fluorochromes used in this protocol on each axis. Gate on the CD3-negative, CD14-positive population.
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